Last year's Cheltenham Gold Cup winner Sizing John has been ruled out of next week's race after suffering an injury on Thursday.
The eight-year-old beat Minella Rocco and Native River under Robbie Power 12 months ago, but will not take on his rivals again as trainer Jessica Harrington announced that the horse had suffered a hairline fracture.
The Nicky Henderson-trained King George VI Chase winner Might Bite is the 3-1 favourite for the March 16 race, while Sizing John had been around 6-1 as second-favourite.
"It is with a heavy heart I have to report that Sizing John will miss the Cheltenham Gold Cup," Harrington told Unibet.
"He cantered as normal this morning at Commonstown and during routine checks this afternoon was found to be sore behind."
Jockey Power was riding in the race for the first time last year when taking victory.
"Heartbreaking to get the call from this evening with the news the Sizing John won't return to challenge for his Gold Cup next week," he wrote on Twitter.
"We're all very disappointed but don't worry, he'll be back!"
Sizing John was described as "clinically abnormal" by a vet after finishing a disappointing seventh behind Road To Respect when odds-on favourite for his last run in Leopardstown's Christmas Chase in December.
The news has seen the odds for several runners slashed, including Native River and Road To Respect.
Earlier on Thursday, Irish jockey Ruby Walsh returned from a four-month injury layoff with victory on favourite Lareena at Thurles in Ireland, just days before the Cheltenham Festival gets underway.
The 38-year-old broke his leg at Punchestown last November, and saw his return delayed by bad weather last weekend.
Walsh, who has won more races at the Cheltenham Festival than any other rider, cruised to a five-length win in the maiden hurdle, beating 6-4 second-favourite Awayinthewest.
He will partner former winner Faugheen in the Champion Hurdle at Cheltenham on Tuesday, looking to topple odds-on favourite Buveur d'Air.